The study focuses on assessing the effect of impurities present in crude glycerol on hydrogen production efficiency. Crude glycerol, a by-product of biodiesel production, is considered a viable carbon source for bio-hydrogen production due to its high energy content. Impurities such as methanol and soap in crude glycerol can significantly influence the yield of hydrogen production. The research employed response surface methodology (RSM) and artificial neural network (ANN) approaches to optimize the impact of glycerol, methanol, and soap concentrations on hydrogen production through dark fermentation. The models predicted a maximum hydrogen yield of 17.67 mmol/L, demonstrating the effectiveness of the optimization techniques. Results indicated that higher initial concentrations of methanol and soap, combined with glycerol, negatively impact hydrogen production efficiency. By conducting controlled experiments, it was found that the presence of impurities in crude glycerol could reduce cumulative hydrogen production by 5%–17% over five days compared to the control. The study emphasizes the significance of exploring impure crude glycerol as a sustainable carbon source for bio-hydrogen production and the necessity to address impurity-related challenges for enhancing hydrogen yield in bioconversion processes.